Cytomegalovirus (CMV) disease is a relatively frequent, and often serious, complication in immunocompromised, CMV-infected patients. In the past few years, it has become apparent that to differentiate between subclinical viral shedding and large-scale viral replication, occurring during the prodrome before the onset of active disease, it is necessary to use sequential monitoring with a quantitative assay. Several studies have shown that CMV quantitative polymerase chain reaction (PCR) assays are more sensitive than buffy coat CMV antigen detection assays. This extra sensitivity can, in some cases, give an additional week of warning before the onset of CMV disease. Instituting antiviral therapy at an earlier time point in the prodromal stage may decrease the chance of the patient developing active CMV disease. We have completed development of a competitive quantitative PCR assay for the detection of CMV in buffy coat cells. The assay can detect as few as three to five viral genome equivalents in an amplification reaction tube. The coefficient of variance of this assay is about 40 percent, in line with other published descriptions of assays of this type. To have an assay with improved precision and, therefore, better potential predictive value for disease onset or progression, we have developed a real-time CMV PCR assay. This assay utilizes frequency resonance energy transfer fluorescence probes and is designed to run on the Roche LightCycler. Amplification and detection of the assay can be completed within 45 to 50 minutes. In addition to continuing the developmental work on a real-time PCR assay, we have begun performing an evaluation of the Organon Teknika NASBA pp67 CMV assay. This commercially available assay amplifies CMV RNA in an isothermal reaction using reverse transcriptase and RNA polymerase. By targeting viral mRNA, it should permit detection of replicating virus without the generating positive signal from integrated viral DNA copies that may be present. We have collected sequential blood sample sets from several patients for an evaluation of the NASBA pp67 CMV assay performance against previously obtained pp65 CMV antigenemia results. We also will use the LightCycler real-time PCR to test these sample sets so that we can compare its performance with the other two methods.